Epidemiological characteristics and structural-functional implications of spike protein variations in a bovine coronavirus isolate from diarrheic calves

Figure 1.. Flow diagram of literature search and selection.

Figure 2.. Detection of BCoV and co-infection patterns of enteric viruses in diarrheic calf samples. (a) Detection of BCoV in 298 diarrheic calf samples by PCR. (b) Co-infection patterns among seven diarrhea-associated bovine viruses in 298 diarrheic calf samples.

Figure 3.. Isolation and characterization of the BCoV strain DDFX98. (a) PCR detection of BCoV following three blind passages in MDBK cells. Lane M, DL2000 DNA marker; lanes 1–3, MDBK cell lysates from blind passages 1–3; lane 4, negative control. (b) PCR detection of BCoV following three blind passages in HRT cells. Lane M, DL2000 DNA marker; lane 1, negative control; lanes 2–4, HRT cell lysates from blind passages 1–3. (c) Transmission electron microscopy of BCoV particles in MDBK cell supernatants. (d) Indirect immunofluorescence assay confirming BCoV infection in MDBK cells.

Figure 4.. Phylogenetic analysis of the complete genome and S gene of the DDFX98 strain. (a) Whole-genome phylogenetic analysis of DDFX98 and 175 representative BCoV strains from different geographic regions, showing classification into three subtypes (GIa, GIb, and GIIb). The DDFX98 isolate is highlighted by a red box, and the contemporary Chinese lineage is highlighted by red branches. The geographic origin of each strain is indicated by the outer color strip. The branches highlighted in red represent the clade comprising Chinese isolates. (b) S gene-based phylogenetic analysis of DDFX98 and 235 representative BCoV strains from diverse geographic origins, indicating four subtypes (GIa, GIb, GIIa, and GIIb). The DDFX98 isolate is highlighted by a red box, and the contemporary Chinese lineage is highlighted by red branches. The geographic origin of each strain is indicated by the outer color strip. The branches highlighted in red represent the clade comprising Chinese isolates. (c) Normalized nucleotide identity heatmaps based on the complete genome and S gene sequences of BCoV strains.

Figure 5.. Comparative structural analysis of the S protein of the DDFX98 isolate and six representative BCoV strains. (a) Multiple sequence alignment of the S1 subunit showing the predicted secondary structural elements and amino acid substitution. (b) Multiple sequence alignment of the S1 subunit showing the predicted secondary structural elements and amino acid substitutions. (c) Schematic representation of the unique amino acid substitutions identified in the DDFX98 S protein. Amino acid substitutions are color-coded as follows: residues unique to DDFX98 are shown in red, substitutions shared with contemporary Chinese isolates are shown in green, and substitutions commonly observed among Asian isolates are shown in blue.

Figure 6.. Structural modeling and conformational comparison of the DDFX98 S protein. (a) Predicted three-dimensional structure of the DDFX98 S protein based on SWISS-MODEL using human coronavirus OC43 (SMTL ID: 9blk.1) as the template. (b) Structural superposition of the DDFX98 and reference strain U00735 spike proteins. Regions exhibiting localized conformational differences are highlighted by red dashed boxes, corresponding to residues 148, 509, and 769. (c – e) enlarged views of the three regions showing localized conformational differences surrounding residues 148, 509, and 769, respectively. Ribbon and surface representations are shown to illustrate the structural changes associated with the corresponding amino acid substitutions. Throughout the figure, the DDFX98 structure is shown in red, whereas the U00735 structure is shown in gree.

Figure 7.. Effects of amino acid substitutions on functional features of the DDFX98 S protein. (a) Prediction of potential N-linked glycosylation sites in the S proteins of DDFX98 and representative BCoV strains using the NetNGlyc server. The red box highlights the glycosylation site affected by the S362G substitution. (b) Prediction of the S1/S2 furin cleavage site in the DDFX98 and reference strain U00735 S proteins using ProP 1.0. The red box indicates the predicted furin cleavage region surrounding the A769S substitution. (c) Predicted transmembrane topology of the DDFX98 S protein generated using DeepTMHMM. The predicted transmembrane region is shown together with the corresponding topology probabilities.